Polypeptide

ABSTRACT

The present invention provides a polypeptide inducing the production of an antibody in permucosal administration in the presence of no immunological adjuvant, a composition containing the polypeptide, and use thereof. The present invention solves the above object by providing a polypeptide inserted with a cell attachment motif of a cell adhesive molecule to the peptide which has an amino acid sequence of multiagretope type T cell epitope at the amino terminal side of an inserted linker peptide, and an amino acid sequence of a B cell epitope at the carboxyl terminal side of the inserted linker peptide, a composition containing the polypeptide, and use thereof.

TECHNICAL FIELD

The present invention relates to a polypeptide having an antigenicepitope, which efficiently induces the production of an antibodyspecific to the antigenic epitope when permucosally administered even inthe absence of an immunological adjuvant, a composition comprising thepolypeptide, and use thereof, more particularly, to a polypeptidedesigned by linking a polypeptide comprising a B cell epitope of aprotein as an antigen for the production of an antibody specific to theprotein to a T cell epitope with a linker peptide comprising amino acidsequence of a protease recognition site, and further linking a peptidecomprising an amino acid sequence of a cell attachment motif, whichefficiently induces the production of an objective antibody, and acomposition comprising the polypeptide, and use thereof.

BACKGROUND ART

In order to induce the production of an objective antibody specific toan antigen in living bodies, a purified antigen has been conventionallyadministered alone or along with an immunological adjuvant (which meansa substance enhancing the immunological reaction specific to an antigenby non-specific stimulation of immune system) to living bodies. Suchmethod has frequently used for preventing various infectious diseases,and been considered as the most effective method because it is alsoeffective even in the case of treating antibiotic-resistant diseasessuch as diseases caused by viruses or toxins produced frommicroorganisms. Particularly, in such case, the method thought to be theonly effective treatment because it enables to defend the living bodiesfrom such diseases by inducing the production of objective antibodiesspecific to viruses and toxins. At present, the method is practicallyapplied to produce vaccines including inactivated vaccines (inactivatedmicrobes without infecting activity, which are prepared by treating withan organic solvent or irradiating UV) and attenuated vaccines(attenuated pathogens with a weak pathogenicity against living bodies).The almost vaccines (except polio vaccines) have been parenterallyadministered by injection. Some inactivated vaccines, however, haveproblems of significant affection to living bodies and less conveniencebecause they require successive injection for several times to obtainsufficient antibody titer to attain a satisfactory biophylaxis.

While, live vaccines such as the attenuated vaccines have problems thatthey have a low preservation-stability and a possibility to becomeharmful virulent mutants after administered to affect living bodies.Both of the inactivated vaccines and the live vaccines must be concernedto be unsafe because they may give an anaphylactic shock to livingbodies. Further, human serum albumin or gelatin has been conventionallyused as a stabilizer and often added to vaccine preparations containinginstable virus particles or huge sized proteins as immunogens to improvethe stability of the vaccine preparations. However, such substances mustbe also concerned to be unsafe because they possibly bring unknowninfectious microorganisms or anaphylactic shock. Most of the vaccinepreparations have been produced for injection use. However, vaccinepreparations have been required to be more stable and applicable withoutinjection in order to be commercialized widely all over the developingcountries where many people are dying of infectious diseases.

To solve the above problems, new type vaccines, having higher safenessand efficacy than conventional inactivated vaccines or live vaccines,has been eagerly studied all over the world by using a polypeptidecomprising a partial amino acid sequence of an inactivated vaccine orlive vaccine. Such new type vaccines are developed as B type hepatitisviral vaccines such as a recombinant vaccine and component vaccine.Peptides used as new type vaccines are usually desired to be a shortfragment. However, excessively short fragments are difficult to bedesigned to have a broad major histocompatibility antigen (hereinafter,abbreviated as “MHC”) class II restriction and satisfactoryimmunogenicity. While, in a view of enhancing immunogenicity of anobjective antigen by permucosal administration, Lavelle, E. C. proposedin Immunology, Vol. 99, pp. 30-37, (2000) an immunizing method of anantigen with an immunological adjuvant such as cholera toxin(abbreviated as “CT”, hereinafter), heat-instable type of enterotoxinfrom E. coli, or attenuated proteins thereof prepared by replacing apart of the amino acid sequence of CT or enterotoxin. The method enablesto obtain a sufficient induction of the objective antibodies. However ithas not been practical because it undesirably brings the induction ofantibodies specific to CT and heat-instable type of enterotoxin from E.coli as immunological adjuvants.

Dental caries and periodontal diseases are considered as the two majordiseases caused by microorganisms in dental field. Since such diseasesare universal and less critical, they should be prevented and treatedunder the highest safeness. As examples of the methods for preventingdental caries, Japanese Patent Publication Kokai No. 122,633/94discloses a passive immunizing method using an antibody obtained from ananimal immunized with a peptide fragment of a tooth surface adhesiveprotein from Streptococcus mutans, and Japanese Patent Publication KokaiNo. 511,422/02 discloses an anti-dental caries vaccine composed of a Tcell epitope and a B cell epitope of glucan binding protein fromStreptococcus mutans.

The present inventor had studied for the purpose of developing a shortpolypeptide which efficiently induces an antibody specific to cellsurface protein antigen from Streptococcus mutans serotype C strain (Itis abbreviated to “PAc”, hereinafter.), which relates to early phaseadhesion to the tooth surface, for many individuals having different MHCclass II haplotype by permucosal administration even in the absence ofimmunological adjuvant. In the detail field, for the purpose ofenhancing the production of antibodies capable of preventing dentalcaries, the inventor proposed to design the short polypeptide in themanner of linearly linking a T cell epitope having multiple restrictionsfrom several MHC class II haplotypes to N-terminus of the partial aminoacid sequence of PAc at the positions of 365 to 377 (SEQ ID NO:1) as a Bcell epitope with a dipeptide linker such as a lysine-lysine sequence(Nishizawa. T., Imai, S., and Hanada, N., Japanese Vaccine conferenceprogram abstract, p. 77, (2000), Oishi Y. et al., Oral Microbiology andImmunology, Vol. 16, pp. 40-44, (2001)). However, even the polypeptidedisclosed in the above reference does not have enough immunogenicity toinduce the production of the antibody. Therefore, the above problems ofconventional peptide vaccines have not still been solved. JapanesePatent Publication Kokai No. 504,118/96 discloses a synthesized peptidevaccine enabling to prevent infection of Chlamydia trachomatis, whichdesigned by linking a B cell epitope to the carboxyl terminal side of aT cell epitope. However, the vaccine did not satisfactorily induce theproduction of the antibody when permucosally administered.

Under the above circumstance, the object of the present invention is toprovide a peptide vaccine, which satisfactorily induces the productionof an antibody in living bodies and safely applicable to living bodieseven when permucosally administered.

DISCLOSURE OF THE INVENTION

The present inventor has studied a polypeptide enabling to more stronglyinduce the production of an antibody, which effectively preventsinfection of dental caries causing microorganisms by a model systemusing a peptide fragment of PAc from Streptococcus mutans known as oneof the microorganisms to solve the above problems.

The present invention is to provide a polypeptide designed by linking acell attachment motif of cellular adhesive molecule to the peptidehaving a T-cell epitope at the amino terminal side of the linker peptideand a B-cell epitope at the carboxyl terminal side of the linker peptideto solve the above problems. As a result, the present inventor foundthat a polypeptide, which comprises (i) an amino acid sequence in theN-terminal region of a peptide, comprising a T cell epitope, restrictedby MHC class II haplotypes in whole; (ii) another amino acid sequencecomprising a B cell epitope for inducing antibody in the C-terminalregion of the peptide, which is located in the peptide via an amino acidsequence, as a linker peptide, recognized by a protease, which isinserted between the two amino acid sequences (i) and (ii); and apeptide comprising an amino acid sequence(s) homologous to a cellattachment motif(s), which is linked to the peptide; and found that thepolypeptide is one with a relatively high safeness, which sufficientlyinduces an antibody specific to the aimed antigen in terms of MHC classII haplotypes, less forms antibodies other than the aimed one, and dosenot substantially induce side effects such as anaphylaxis whenpermucosally administered, for example, in an intranasal manner in theabsence of immunological adjuvant.

The present invention provides a polypeptide designed by inserting acell attachment motif of a cellular adhesive molecule to a peptidehaving a T cell epitope at the N-terminus of the linker peptide and Bcell epitope at the C-terminus of the linker peptide to solve the aboveproblems.

THE BEST MODE FOR CARRYING OUT THE INVENTION

The term “antibody” as referred to as in the present invention meansimmunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A(IgA), and includes those secreted intranasally, orally, orbitally andintestinally as well as in the blood and the fluids body.

The B cell epitope sequence in the polypeptide of the present inventionconsists of an amino acid sequence of B cell epitope and optional othersequences to induce objective antibodies. It can be freely selected fromB cell epitopes of various antigens such as toxins, allergens, enzymes,cell surface antigens, and tumor specific antigens from microorganisms,normal cells, and tumor cells. It also can be selected from antigenicepitopes disclosed literally or identified newly by a usual immunizingmethod using a partial peptide as long as it actually exertsantigenicity against living bodies. It can be selected from the sameantigens having T cell epitopes (described below) used in thepolypeptide of the present invention. In addition, it can be partiallyor wholly shared by the T cell epitopes. Therefore, any amino acidsequence having a B cell epitope can be freely selected from antigenicpolypeptides, varying depending on the purposes of the objectivelyinduced antibodies. Such purposes are examples of prevention ofinfection; prevention or treatment of cancers, tumors, ulcers,inflammatory diseases such as hepatitis, immunological diseases such asallergy and atopic dermatitis; neutralization of enzymes; and detectionfor various antigens used in a clinical test or the like. For example,the PAc peptide fragment described above (reported by Nishizawa. T.,Imai, S., and Hanada, N., Japanese Vaccine conference program abstract,p. 77, (2000)) consists of an amino acid sequence of SEQ ID NO:1, andcan be used for inducing antibodies capable of preventing dental caries.The part of B cell epitope sequence is usually composed of single B cellepitope. Desirably, such sequence can be in the form of a dimer, trimeror polymer by linking tandemly with one or more same or different B cellepitopes of the same antigen. In addition, the whole sequence of anantigen having a B cell epitope(s) can be used as the sequence. In orderto enable the polypeptide of the present invention to treat a diseaserelating to some different antigenic proteins even in a single use, thepolypeptide of the present invention can be designed to have atandemly-linked multiple B cell epitopes chosen from the antigens. Suchpolypeptide is capable of inducing the multiple productions ofantibodies. In such case, B cell epitopes in the polypeptide should beseparated from each other with a linker peptide described below in orderto be surely processed.

The T cell epitope sequence in the polypeptide of the present inventioncomprise any T cell epitope as long as it is restricted by an MHC classII haplotype of objective animals such as mammals including humans,fowls, reptiles and fishes, and presented as antigens for helper Tcells. The T cell epitope sequence can be selected from the amino acidsequences literally disclosed as T cell epitopes. In addition, such Tcell epitopes can be newly identified by a usual method, i.e.,immunization with a partial peptide of an antigen, in order to use as aT cell epitope sequence of the polypeptide of the present invention. TheT cell epitopes can also be selected from the same antigens to those ofabove-described B cell epitopes used in the polypeptide of the presentinvention. In addition, it can be partially or wholly shared by the Bcell epitope. The T cell epitope sequence can be composed of a single Tcell epitope or some of same or different T cell epitopes tandemlylinked together. The T cell epitope sequence can exert its function evenif the amino acid residues are substituted with other amino acidresidues as long as agretopes (required for linking with MHC class IIantigen) are conserved. Therefore, any amino acid sequence withconserved agretopes can be used as the T cell epitope sequence in thepresent invention. Multiagretope type polypeptide, which means apolypeptide having tandemly-arranged or overlapped agretopes restrictedby multiple MHC class II haplotypes, can be more advantageously used asthe T cell epitope part of the polypeptide because it enables thepolypeptide to be applicable for many patients in order to defend personfrom infection, or prevent or treat allergic diseases. Many basicstructures or agretopes restricted by MHC class II haplotype aredisclosed literally. Based on such disclosures, the polypeptidecomprising the T cell epitopes can be freely designed to have T cellepitopes suitable for its use. For example, the polypeptides havingtandemly-arranged or overlapped T cell epitope or agretope sequencesselected from that of different kinds of animals are acceptable to somedifferent kinds of animals.

In addition, the B cell epitope sequence described above with thetandemly-arranged or overlapped B cell epitopes can be also used toinduce some kinds of antibodies simultaneously.

If a polypeptide having only T cell epitope sequences and anotherpolypeptide having only B cell epitope sequences are immunized withoutcoupling each other, the production of objective antibodies is notefficiently enhanced. While, if the polypeptides is produced in a manerof simply couplig the B cell epitope sequence and the T cell epitopesequence, it possibly induces undesired antibodies recognizing a linkingpart with the desired antibodies simultaneously. Therefore, thepolypeptide of the present invention is designed to inhibit theproduction of the antibodies recognizing the above amino acid sequenceby inserting the linker peptide comprising the amino acid sequence of aprotease recognition site for the purpose of allowing the polypeptide tobe enzymatically divided into two polypeptides through the processingstep of antigen presentation. Therefore, the polypeptide of the presentinvention has satisfactory immunogenicity, and enables to selectivelyenhance the production of objective antibodies specific to the B cellepitope sequence in a sufficient amount to exhibit the defending effecton living bodies without enhancing antibodies specific to thepolypeptide.

The linker peptide used in the polypeptide of the present invention isinserted between the B cell epitope sequence and the T cell epitopesequence to couple them. Such linker peptides can be selected fromprotease recognition site sequences relating to antigen processing. Suchsequences are examples of lysine-lysine (KK) dipeptide, lysine-arginine(KR) dipeptide and arginine-arginine (RR) dipeptide. Particularly,lysine-lysine dipeptide, which is a cathepsin B recognition site, ismore desirable. If two polypeptides having a sequence overlapped T cellepitopes with B cell epitopes are linked together with a linker peptide,the resulting polypeptide enables to exert the both functions of T cellepitope and B cell epitope.

The cell attachment motif of cellular adhesive molecule used in thepolypeptide of the present invention has a function to allow thepolypeptide to stay on the surface of the mucosal cells for a longperiod. It can be selected from amino acid sequences of cell attachmentmotifs of cellular adhesive molecule including integrin family proteinsor others. Examples of the amino acid sequences of integrin bindingmotif are arginine-glycine-asparagic acid (RGD: SEQ ID NO:2),arginine-glutamic acid-asparagic acid (RED: SEQ ID NO:3),leucine-asparagic acid-valine (LDV: SEQ ID NO:4),proline-histidine-serine-arginine-asparagine (PHSRN: SEQ ID NO:5),arginine-lysine-lysine (RKK: SEQ ID NO:6), and asparagicacid-glycine-glutamic acid-alanine (DGEA: SEQ ID NO:7) which are knownas a cell attachment motif existing on cellular adhesive molecules suchas fibronectin, collagen, vitronectin, fibrinogen, laminin, Tat proteinof human immunodeficiency virus (abbreviated as “HIV”, hereinafter).Examples of the amino acid sequences of the cell attachment motifswithout belonging among integrin family aretyrosine-isoleucine-glycine-serine-arginine (YIGSR: SEQ ID NO:8),isoleucine-lysine-valine-alanine-valine (IKVAV: SEQ ID NO:9),arginine-phenylalanine-tyrosine-valine-valine-methionine-tryptophane-lysine(RFYVVMWK: SEQ ID NO:10), andisoleucine-arginine-valine-valine-methionine (IRVVM: SEQ ID NO:11).Particularly, RGD, RED, or YIGSR are desirable because they enhance theproduction of specific antibodies more strongly. The cell attachmentmotifs can be inserted at one or more positions selected from N- andC-terminus of the T cell epitope sequence and the B cell epitopesequence. They are preferably inserted into the polypeptide atN-terminus and/or C-terminus of T cell epitope sequence because ofenabling to more strongly induce the production of antibodies specificto antigens having the B cell epitope sequence.

The method for producing the polypeptide of the present invention is notspecifically restricted. The polypeptide can be prepared by a usualpeptide synthesizing method. Optionally, the synthesized peptides can belinked each other according to The Japanese Biochemical SocietiesCorporation, Sin-Seikagaku-Jikken-kouza (New biochemical experimentalCourse), Vol. 1, “Tanpakushitsu (Protein) VI”, pp. 3-44, (1992),published by Tokyo Kagaku Dojin Co., Ltd., Tokyo, Japan). The peptidecan be synthesized using a peptide synthesizer commercialized by anymanufacturers according to the attached protocols. The polypeptide canbe also prepared according to conventional recombinant DNA techniquescomprising the steps of; preparing a DNA coding for an amino acidsequence of the designed polypeptide; inserting the DNA into aself-replicable vector and introducing the resultant into a host, i.e.,microbes such as E. coli, Bacillus subtilis, Actinomycete, and yeast,animals, plants, cells or tissues thereof; or generating the transgenicanimals or plants; culturing or breeding the resultants; and collectingor purifying the polypeptide of the present invention by a usual method.Optionally, the polypeptide can be prepared by connecting thepolypeptide with digestion sites of proteases other than that ofdigestion sites used for linking a T cell epitope sequence to a B cellepitope sequence, expressing the resulting polypeptide, and digestingthe resulting polypeptides with the proteases. The microbes, animals, orplants having the expressed polypeptides of the present invention can befreely used intact as a composition comprising the polypeptide for oraluse. The polypeptide can be directly prepared according to the abovemethods. In addition, It can be prepared by chemically binging thepolypeptides prepared according to the above methods each other.

The polypeptide can be used alone or in combination with somepolypeptides having the same B cell epitope. In order to simultaneouslyproduce antibodies specific to several antigens, the polypeptides of thepresent invention can be freely used in combination with at least two ofthem having different B cell epitopes corresponding to each antigen. Acomposition comprising one or more pharmaceutically acceptable additivescan be advantageously prepared as long as it does not affect the effectof the present invention. Examples of such additives are reducing ornon-reducing saccharides such as glucose, maltose, trehalose, andsucrose; sugar alcohols such as sorbitol, mannitol, and maltitol;aqueous polymers such as agar, pullulan, guar gum, and gum arabic;proteins such as gelatin and silk, and hydrolysates thereof; effectiveingredients of nutrition products with health promoting benefits,medical cosmetics, and pharmaceuticals such as lipids, amino acids,buffers, stabilizers, antimicrobials, and perfumeries; and immunologicaladjuvants such as alum and aluminum hydroxide. One or more of the aboveingredients can be used alone or in combination. The form of thepreparations comprising the polypeptide of the present invention is notrestricted to a specific one as long as it is stably preserved for along period. It can be freely selected from a solution, freeze-driedproduct, tablet, baccal, lozenge, powder, granule, cream, ointment, andsyrup, varying depending on patient, administration, preservation, andtransportation. The polypeptide of the present invention or thecomposition as above can be included in a liposome. It can beadvantageously used in combination with an accelerator for facilitatingthe penetration into skins or tissues, and applied by iontophoresis tofacilitate the penetration into the part with antigen presenting cells.Various foods and beverages such as a tablet candy, candy, and softdrink, which comprise the polypeptide of the present invention, can beorally intaken to allow the polypeptide to be absorbed permucosally. Thepolypeptide can be allowed to express in living bodies by gene therapy,i.e., direct administration of an RNA coding for the polypeptide of thepresent invention or by introduction of a DNA coding for the polypeptideto cells.

The polypeptide allows antibody productive animals such as mammalsincluding humans, dogs, cats, or mice, fowls including chickens andducks, reptiles, and fishes particularly hatchery fishes to produceantibodies to constitutive proteins or toxins which are inherent to orsecreted from pathogenic virus, microbes, or bacteria. Therefore, thepolypeptide effectively prevents or treats food poisonings caused bybacteria such as bothulinum and E. coli OH-157, and infectious diseasessuch as tetanus, diphtheria, and influenza. The polypeptide alsoeffectively treats Alzheimer's disease which produces antibodies toamyloid β peptide, or it can be advantageously used in oralhyposensitization therapy for various allergy diseases or atopicdermatitis by predominantly producing specific IgG antibodies toallergens of mites, house dusts, pollens, foods, etc. The polypeptide ofthe present invention induces the production of IgA on mucosae and IgGin body fluids by permucosally administering into oral mucosae, nasalmucosae, orbital mucosae, guttural mucosae, vaginal mucosae,endotracheal mucosae, peritoneal mucosae, pleural mucosae, alveolarmucosae, esophagal mucosae, or alimentary mucosae such as gastricmucosae and intestinal mucosae. In addition, it can be administeredsubcutaneously, intracutaneously, or intramuscularly similarly inconventional vaccines, or in some cases it can be administeredintravascularly. The polypeptide is useful for the production ofspecific antibodies to any proteins and intrinsically for antibodyproduction against low-antigenic oligopeptides or polypeptides.Therefore, it can be advantageously used in such a manner of immunizinganimals with synthesized oligopeptides for the purpose of producingantisera, and sensitizing immunocompetent cells in vitro as well asimmunizing animal with synthesized oligopeptides for the purpose ofproducing monoclonal antibodies.

The polypeptide is also useful as an immunological adjuvant whichinduces the production of an antibody to be simultaneously administeredwith an antigen when administered to living bodies in combination withother antigen(s) or an antigenic protein deriving a B cell epitope ofthe polypeptide. Dose of the administration of the polypeptide is notrestricted to a specific one as long as it sufficiently induces theproduction of an antibody to other antigen administered simultaneouslyor an antigen deriving the B cell epitope of the polypeptide. When theinduction of the production of antibody to the polypeptide of thepresent invention is not desired, the dose of the polypeptide is usually10% by weight or less, preferably, 1% by weight or less, morepreferably, 0.1% by weight or less to the other antigen(s).

The administration method of the polypeptide of the present invention isnot restricted to a specific one as long as it surely transports thepolypeptide to the desired position. For example, it can be dropped onmucosae with a dropper or syringe, ingested orally, applied on mucosaeafter formed in a cream or gel form, introduced into the desiredposition with a catheter, sprayed after formed in a mist form with aspray or nebulizer, or aspirated into the nose, trachea, or lung. Anadministration method using a syringe, catheter, or intravenous drip canbe used when the polypeptide is administered subcutaneously,intracutaneously, intramuscularly, intravascularly, and intracoelarlysuch as intraperitoneally and intrapleurally. Varying depending on theactivity of inducing antibody, kind of disease, administration pathway,administration method, animal to be treated/prevented, a dose of thepolypeptide of the present invention is usually 0.00001 to 100 mg/kgbody weight, preferably, 0.0001 to 25 mg/kg body weight, morepreferably, 0.001 to 10 mg/kg body weight.

The following experiments explain the polypeptide of the presentinvention in more detail.

EXPERIMENT

For the purpose of developing the polypeptide usable as a vaccine fordefending living bodies from infectious disease, the followingexperiments were carried out to obtain vaccine polypeptides enabling toefficiently induce the production of antibodies in a permucosaladministration using a model system of the production antibodiesspecific to PAc (a bacterial surface protein antigen as of toothadhesive factors) of Streptococcus mutans serotype C strain (a kind ofpathogen causing dental caries). The amino acid sequence of SEQ ID NO:1in the following experiments is known to have cross-reactivity to PAcmolecule and be minimum unit peptide antigen (It is described as “unitpeptide” abbreviated to “UP”, hereinafter) that induces only antibodyhaving antidental caries activity (reported by Senpuku H., Infection andImmunity, Vol. 63, pp. 4695-4703). Polypeptides used in followingexperiments were synthesized by Fmoc method using a peptide synthesizer(“Model 350 Multiple Peptide Synthesizer” commercialized by AdvancedChemtech Corporation), and purified up to 95% or over by reverse phaseHPLC using “TSK-GEL” a column sized 1 cm in diameter and 30 cm inlength, commercialized by Tohso Co., Ltd., Tokyo, Japan.

Experiment 1

Analysis of Restriction of Mouse MHC Class II Haplotype Against UP

Five-week aged B10 congenic female mice, commercialized by Japan SLC Co.Ltd., Tokyo, Japan, were divided into eight groups consisting of fivemice in view of difference of MHC class II haplotype. The all mice wereintraperitoneally immunized with 200 μl/head of the UP and Freund'sincomplete adjuvant (It is abbreviated as “FIA”, hereinafter.), andboosted with them under the same condition after two weeks. All the micewere bled after one week. And then, the resulting antisera weresubjected to measuring the titer against UP or PAc by a usual ELISAmethod using the UP or PAc as a coating antigen. The titers against theantigens are measured as follows; the antisera prepared of mice wereserially diluted twofold and placed in microtiter plates to be subjectedto measuring amount of antibodies by ELISA method using enzyme-labeledantibodies, and then, the resulting plates were subjected to measuringabsorbance in 405 nm of each well using “MULTISCAN Bichromatech”, amicrotiter plate reader commercialized by Labosystem Corporation. Thevalues of titers were calculated by averaging the maximum dilutionvalues showing that the difference between the resulting values of wellcoated with the antigen and that of well uncoated one is 0.1 or over.The result is shown in Table 1. The parenthesized values followed eachname of congenic mouse mean MHC class II haplotype.

TABLE 1 Congenic MHC class II Titer Titer mice haplotype against PAcagainst UP B10.M f 19,112 35,391 B10.D2 d 1,625 9,314 B10.BR k 1,1354,539 B10.SM v 138 75 B10.S s 138 75 B10.Y pa 75 75 B10.G q 75 45B10.RIII r 40 25

As evident from the result in Table 1, B10.M, B10.D2, and B10.BRcongenic mice having MHC class II (H-2) haplotype f, d, and k,respectively, demonstrated that the production of specific antibody toUP and PAc is more strongly induced than other mice did (over abouttenfold). Therefore, the UP was revealed to have effective B cellepitope capable of inducing specific antibodies and effective multiple Tcell epitopes essential for antigen presentation on congenic mice havingeither MHC class II (H-2) haplotype of d, f or k. Since the UP isresponsible to some mice having a different haplotype of MHC class II(H-2) in spite of a short peptide consisting of just 13 amino acidresidues, it is considered to be a multiagretope type of T cell epitope.The result shows that the polypeptide of the present invention, even ifbeing single and short, is possible to be artificially designed tosufficiently induce the production of antibodies in some individualshaving a different haplotype of MHC class II.

Experiment 2

Confirmation of Multiagretope Type T Cell Epitope of Mouse

The above Experiment 1 confirmed presence of multiagretope type T cellepitope in the UP. Following experiment was carried out to confirm thelocation of functional amino acid. Valine substituted peptides weresynthesized in the manner of exhaustively substituting any one of aminoacid residue of UP with one valine residue, and subjected to immunizingthe responsive mice to UP described above. As a result, the amino acidresidues essential to induce the production of the antibodies in eachmouse were deduced. Following, peptides substituted with valine residueswere synthesized in the manner of substituting all of the deduced aminoacid residues of UP with one valine residue, and subjected to immunizingaccording to the above. The resulting antisera were subjected tomeasuring their titers using ELISA. The agretopes of the UP responsibleto each MHC class II haplotype were determined by analyzing theefficiency of the production of antibodies. The results are shown inTable 2 all together. The Table shows the only amino acids recognized byMHC class II haplotype.

TABLE 2 1 2 3 4 5 6 7 8 9 10 11 12 13 Amino Acid Sequence of Thr Tyr GluAla Ala Leu Lys Gln Tyr Glu Aal Asp Leu UP Amino Acid relating to AlaLys Gln Ala restriction of B10.A(a) Amino Acid relating to Glu Leu LysAsp Leu restriction of B10.D2(d) Amino Acid relating to Leu Tyr Glu Leurestriction of B10.M(f) Amino Acid relating to Thr Tyr Ala Glnrestriction of B10.BR(k)

As evident from the result in Table 2, the polypeptide consisting of 13amino acid residues has amino acid residues essential for recognition ofMHC class II haplotype (agretope) in the overlapped or shifted form. Thelocation of amino acid recognized by each MHC class II haplotype wasconfirmed as a multiagretope type T cell epitope in the UP.

Experiment 3

Design of Multiagretope Type Peptide Antigen

It is investigated whether remodeling the above UP by substituting theiramino acids is possible to change immunological response relating torestriction of MHC class II haplotype in mice or not. The peptide havingamino acid sequence of SEQ ID NO:12 (described as “YQTEL peptide”,hereinafter), SEQ ID NO:13 (described as “YETDL peptide”, hereinafter),and SEQ ID NO:14 (described as “YETAL peptide”, hereinafter), which aredesigned by changing the part corresponding to agretopes of B10.D2(d)and B10.M(f) mouse (at the position of 10 to 12 of the amino acidsequence of SEQ ID NO:1) determined in Experiment 2, amino acid sequenceof SEQ ID NO:1 (described as “YEADL peptide”, hereinafter), SEQ ID NO:15(described as “YEADLKQY peptide”, hereinafter), which is designed byadding lysine-glutamine-tyrosine to the amino acid sequence of SEQ IDNO:1 at the carboxyl terminal, SEQ ID NO:17 (described as“UP-PAc(305-318)”, hereinafter), which is designed by linking thepeptide of SEQ ID NO:16 having the amino acid sequence at the positionsof 305 to 318 from amino terminal of PAc known as a T cell epitoperecognized by B10.S(s) mouse (described as “PAc(305-318), hereinafter”)to amino terminal of UP, and SEQ ID NO:18 (described as“PAc(305-318)-UP”, hereinafter), which is designed linking the UP andPAc(305-318) in reverse were synthesized and applied to B10 congenicmice to be immunized by the same method and schedule to Experiment 1.After two weeks from the boosting immunization, the antisera wereprepared from the resulting blood samples, diluted with physiologicalsaline 512-fold, and subjected to measuring the production of theantibodies by ELISA method according to Experiment 1. The production ofantibodies in each congenic mouse was measured absorbance in 405 nm tobe compared. The result was shown in Table 3. The result of measuringthe production of antibodies specific to UP in mouse immunized withUP-PAc(305-318) and PAc(305-318)-UP were shown in Table 4. The antiserumpresented 0.1 or more absorbance was judged to have antibody against UPin the diluted serum.

TABLE 3 MHC class Titer against UP (Absorbance in 405 nm) Congenic IIYQTEL YETDL YETAL YEADL YQADLKQY PAc(305- mouse haplotype peptidepeptide peptide Peptide Peptide 318)-UP B10.A a 0.01 0.01 0.01 0.28 0.290.29 B10.D2 d 0.01 0.32 0.32 0.33 0.32 0.30 B10.M f 0.01 0.12 0.33 0.320.32 0.32 B10.BR k 0.22 0.31 0.30 0.30 0.30 0.30 B10.S s 0.01 0.01 0.010.01 0.01 0.31 B10.SM v 0.01 0.01 0.01 0.01 0.32 0.31

TABLE 4 Titer Against The UP Congenic MHC class II Immunizing withImmunizing with mouse haplotype UP-PAc(305-318) PAc(305-318)-UP B10.D2 d7,610 28,695 B10.S s 106 108,205 B10.M f 59,986 108,205

As evident from the result in Table 3, immunizing with YQTEL peptideartificially designed form UP induced the production of the antibody foronly one B10.BR(k), while immunizing with YEADL peptide (UP)simultaneously induced the production of the antibody for three kinds ofmice except of B10.S(s) and B10.SM(v). It is confirmed that changing oneto three amino acids is effective to change the immunologicalresponsibility for different individuals in view of MHC class IIhaplotype. YEADLKQY peptide, which is designed by addinglysine-glutamine-tyrosine to the UP, induced the production of antibodyfor four kinds of mice other than B10.S(s). Further, as evident from theresults in Table 3 and 4, PAc(305-318)-UP which is designed by linkingPAc(305-318) responded by B10.S(s) mouse irresponsible to the UP to theamino terminal of the UP, was confirmed to be a multiagretope typepeptide antigen capable of inducing the production of the antibody forfive kinds of mice including s type. Therefore, the above resultsrevealed that artificially designing the polypeptide having theoverlapping amino acid sequence restricted by each MHC class IIhaplotype based on the analysis of the amino acid sequence relating tomore than one MHC class II haplotype restrictions on the antigen,enables to construct overlapping type of multiagretope type T cellepitope, which efficiently induces the production of the antibody to theantigen and is simultaneously restricted by more than one MHC class IIhaplotypes. The polypeptide designed by linking more than one T cellepitopes tandemly, so-called as “cluster type T cell epitope”, isconfirmed to be T cell epitope capable of efficiently inducing theproduction of the antibody as well as overlapping type epitope forindividuals having more than one MHC class II haplotype restrictions. Asevident from the result in Table 4, it is observed that the polypeptidedesigned by linking the UP to C-terminal side of PAc(305-318) morestrongly induces the production of antibodies rather than thepolypeptide designed by linking the UP to N-terminal side ofPAc(305-318).

Experiment 4

Artificial Construction for Multiagretope Type Peptide Antigen for HumanUse

Under the above result of experiment 3, in order to solve a problem ofthe individual difference due to MHC class II haplotype restriction, anoverlapping multiagretope peptide (abbreviated as “OMP”, in thefollowing Experiments) having amino acid sequence of SEQ ID NO:19, whichis responsible for more than one class II haplotypes, was constructedbased on some preiously disclosed human restriction motifs of MHC classII haplotype (HLA-DR) against T cell epitopes. The peptide is capable ofexerting for mouse the same function to human as T cell multiagretope(data not shown). Following Table 5 shows the amino acid recognized byeach human MHC class II HLA-DR haplotypes.

TABLE 5 1 2 3 4 5 6 7 8 9 10 Amino Acid Sequence of Leu Ala Val Tyr TrpGlu Leu Leu Ala Lys OMP Amino Acid relating to Tyr Leu Ala restrictionof HLA-DR1 Amino Acid relating to Val Glu restriction of HLA-DR3 AminoAcid relating to restriction of HLA-DR4 Amino Acid relating torestriction of HLA-DR5 Amino Acid relating to restriction of HLA-DR6Amino Acid relating to Tyr Leu restriction of HLA-DR7 Amino Acidrelating to restriction of HLA-DR8 Amino Acid relating to Trp Leu Lysrestriction of HLA-DR11 11 12 13 14 15 16 17 18 19 20 Amino AcidSequence of Tyr Leu Leu Asp Arg Val Gln Lys Val Ala OMP Amino Acidrelating to Leu restriction of HLA-DR1 Amino Acid relating torestriction of HLA-DR3 Amino Acid relating to Tyr Asp Val Glnrestriction of HLA-DR4 Amino Acid relating to Leu Arg Val Lysrestriction of HLA-DR5 Amino Acid relating to Leu Arg Val Lys Alarestriction of HLA-DR6 Amino Acid relating to Leu restriction of HLA-DR7Amino Acid relating to Leu Val Lys restriction of HLA-DR8 Amino Acidrelating to restriction of HLA-DR11

Experiment 5

Influence of Linker Peptide for Enhancing the Production of AntibodiesSpecific to the Polypeptide

The UP prepared in Experiment 3 does not have sufficient immunogenicitydue to a too short peptide. In order to enhance its immunogenicity,tandemly linked polypeptides were tested. However, if the UPs weresimply linked each other, the resulting peptides were concerned to havea new epitope sequence inducible of new antibodies at the linking part.For the purpose of allowing the only desired B cell epitope sequence topresent, a linker peptide was arranged between the T cell epitopesequence and B cell epitope sequence. Such linker peptide is KK, whichis previously disclosed as recognition site of endosome proteasecathepsin B relating to the processing of antigen in antigen presentingcell. Following experiment is to confirm the influence of the linkerpeptide for enhancing the production of antibodies.

The polypeptides designed by inserting with KK between OMP and UP,represented by “OMP-KK-UP” having amino acid sequence of SEQ ID NO:20and “UP-KK-OMP” having amino acid sequence of SEQ ID NO:21,respectively, were synthesized. The OMP was prepared in Experiment 4 andhad a mouse T cell epitope, and the UP comprised a B cell epitope. Theywere immunized to BALB/c mice intraperitoneally by the method accordingto Experiment 1. The serum was prepared from mice and titers againstOMP, the immunized polypeptide, and PAc were measured by the methodaccording to Experiment 1. The result is shown in Table 6.

TABLE 6 Polypeptide Titer against Titer used as an Titer against theImmunized against antigen PAc peptide OMP OMP-KK-UP 836,462 782,6851,731 UP-KK-OMP 961 29,921 1,211

As evident from the result in Table 6, OMP-KK-UP; arranging T cellepitope sequence at N-terminal region and B cell epitope sequence atC-terminal region and inserting a linker peptide between the twosequences; was revealed to efficiently induce the production ofantibodies. This tendency was also observed in other strains of mice.Particularly, such tendency was remarkably observed in B10.S(s) mousewhich PAc(305-318) was functional as a T cell epitope but UP was not so(referred to Table 4). It was revealed that the peptide at N-terminalside of the linker peptide is more effectively recognized as T cellepitope and the peptide at carboxyl terminal of the linker peptide ismore effectively recognized as B cell epitope.

Experiment 6

Effect of a Cell Attachment Motif of Cell Adhesive Molecule on theEnhancement of Intranasal Immunogenicity of a Peptide Antigen

A polypeptide having SEQ ID NO:22, arranged with two molecule of the UP,which has the amino acid sequence of SEQ ID NO:1 and induces theproduction of the antibodies having antidental caries activity, with thelinker, lysine-lysine, (described as “di unit peptide” and abbreviatedas “DUP”, hereinafter) is so efficiently capable of enhancing theproduction of antibodies to need no immunological adjuvant in the caseof injection use (subcutaneously or intraperitoneally). However, in thecase of intranasal immunization, it is insufficient to use singly andactually needs an immunological adjuvant. In order to enhance theintranasal immunogenicity of the DUP, a cell attachment motif, which ispossible to retain the DUP on mucosae for a long period, was introducedin the DUP. Such cell attachment motif was selected from integrinbinding motifs of cell adhesion proteins such as fibronectin, lamininand collagen, which were determined to have a cell adhesion property.The following experiment was carried out to confirm the influence of thecell attachment motif on the immunogenicity. At first, the polypeptideshaving one or more cell attachment motif selected from RGD, RED, LDV,PHSRN, RKK, DGEA, YIGSR, IKVAV, IRVVM, and RFYVVMWK, at the N-terminalregion were synthesized. As a negative control, DUP was prepared. Ascontrols, polypeptides having DUP and either DRE (SEQ ID NO:23), DED(SEQ ID NO:24) or HAV (SEQ ID NO:25), which are amino acid sequencesrelating to cadherin without showing cell adhesion property in singleuse, were synthesized. Five week-aged female BALB/c mice, commercializedby Japan SLC, Inc., Shizuoka, Japan, were intranasally administered with4 μl of phosphate buffer saline (hereinafter, abbreviated as “PBS”) ordistilled water containing 50 μg of each sterilized polypeptide in amanner of dropping by 2 μl into both nostrils. Mice of the negativecontrol were administered with DUP alone. Mice of a positive controlwere administered with the DUP and 1 μl of CT. The DUP and thepolypeptide having DUP and RGD were also intraperitoneally administered.After two weeks from the first immunization, the same polypeptide as abooster was administered again in the same manner. After two weeks fromthe second immunization, the same polypeptide as a booster wasadministered again in the same manner. After one week from the thirdimmunization, sera were prepared from mice and subjected to measuringthe titers of antubodies against PAc, DUP, CT and the polypeptide havingthe DUP and cell attachment motifs using the ELISA method according toExperiment 1. In order to confirm the effect of RGD or YIGSR sequence, apeptide fragment consisting of RGDS (SEQ ID NO:26) or YIGSR (SEQ IDNO:27) as an inhibitor against polypeptides having RGD or YIGSR sequencewas applied together with the polypeptide to the mice in an excessamount to immunize the mice. The results are shown in Table 7. The miceimmunized with the polypeptides having one or more motifs selected fromRGD, RED, LDV, PHSRN, RKK, DGEA, YIGSR, IKVAV, IRVVM and RFYVVMWK showedthe effect on the enhancement of the production of the antibodiesspecific to the UP. While, the mice immunized with the polypeptidehaving DRE, DED or HAV did not show such effect. The results of DUPssuch as SEQ ID NO:27 (“RGD-DUP”, hereinafter), SEQ ID NO:28 (“RED-DUP”,hereinafter), SEQ ID NO:29 (“YIGSR-DUP”, hereinafter), SEQ ID NO:30(“DED-DUP”, hereinafter) and SEQ ID NO:31 (“HAV-DUP”, hereinafter) aretypically shown in Table 7.

TABLE 7 Polypeptide used as an antigen Administration Titer against PAcDUP Intranasal 640 CT + DUP administration 32,768 RGD-DUP 5,069 RGDS +RGD-DUP 256 RED-DUP 1,395 YIGSR-DUP 1,195 YIGSR + YIGSR-DUP 235 DED-DUP544 HAV-DUP 320 DUP Intraperitoneal 312 RGD-DUP administration 2,352

As evident from the result in Table 7, UP was not observed to induce theproduction of antibodies specific to the UP when intranasallyadministered in single use. While, the polypeptide having RGD or REDsequence as integrin attachment motif, or YIGSR as an attachment motifof laminin against laminin binding protein, i.e., RGD-DUP, RED-DUP, orYIGSR-DUP, was observed to have the effect on enhancing the productionof antibodies. Adding an excess amount of RGDS or YIGSR peptidefragment, which is a short peptide as an inhibitor of binding tointegrin, inhibited the production of antibodies specific to the PAc.The result suggested that cell attachment motif such as RGD and YIGSR iseffective on more strongly inducing the production of antibodies, andthe effect exerted by binding the DUP to integrins on cell surface ofmucosae.

Experiment 7

Influence of Cellular Adhesive Molecule for Enhancement of theProduction of Antibodies by Permucosally Administered Peptide Vaccine

Following experiment is to investigate influence for enhancement ofother peptide antigen by addition of integrin binding motif demonstratedin Experiment 6 and due to the position of the motif. Four kinds of thepolypeptides; “RGD-OMP-KK-UP” (SEQ ID NO:32); “OMP-RGD-KK-UP” (SEQ IDNO:33); “OMP-KK-RGD-UP” (SEQ ID NO:34); “RGD-KK-UP-RGD” (SEQ ID NO:35)were synthesized in the manner of introducing RGD sequence into“OMP-KK-UP” at the positions of N-terminal or C-terminal side of the OMPor the UP; Wherein the RGD sequence was a kind of integrin binding motifand is suggested to enhance the production of antibodies specific to thepolypeptide according to Experiment 6, and wherein the “OMP-KK-UP” wasconfirmed to be high enhancement of production of the specific antibodyto PAc according to Experiment 5. As a positive control, CT is used,which is previously disclosed to have an adjuvant activity in permucosaladministration. B10 mice were immunized with solutions containing eachpolypeptide with or without CT according to Experiment 6, and then,effects of each peptide on enhancing the production of antibodies werejudged. The result is shown in Table 8.

TABLE 8 Titer against Polypeptide used Titer the immunized Titer againstas an antibody against PAc peptide OMP OMP-KK-UP 32 128 32 CT +OMP-KK-UP 1,454 1,063 14 RGD-OMP-KK-UP 11,544 11,514 32 OMP-RGD-KK-UP26,241 24,370 16 OMP-KK-RGD-UP 169 723 16 OMP-KK-UP-RGD 1,981 9,483 16

As evident from the result shown in Table 8, the insertion of RGD intoOMP-KK-UP peptide enhanced the production of antibodies specific to PAc.Particularly, in the case of the inserting RGD at the position ofN-terminal or C-terminal side of OMP, in other words, in N-terminal sidefrom the linker, the polypeptide more remarkably enhanced the productionof antibodies rather than the polypeptide having no RGD with CT. Thealmost of produced antibodies were capable of reacting to PAc. While,the polypeptides having RGD in C-terminal side from the linker inducedmany antibodies specific to the peptide including RGD sequence.

From above-described, the sequence of N-terminal side from the linker isrecognized as T cell epitope, and the sequence of C-terminal side fromthe linker is recognized as B cell epitope in the presence of noadjuvant in intranasal immunization. The polypeptide efficientlyenhances the production of antibodies specific to objective B cellepitope sequence. Particularly, it is revealed that the polypeptidehaving RGD sequence in N-terminal side from the linker is capable ofinducing the antibodies specific to only PAc.

Experiment 8

Generality of Basic Design of Peptide Vaccine for PermucosalAdministration

Following experiment is to investigate influence for the production ofantibodies specific to B cell epitope sequence in the case of replacingT cell epitope or B cell epitope of the polypeptide constructed asRGD-(T cell epitope)-KK-(B cell epitope) according to Experiment 7 byplacing other sequence instead of OMP or UP. The polypeptide representedby “RGD-OMP-KK-UP” (SEQ ID NO:38) or “T1-RGD-KK-UP” (SEQ ID NO:39) weredesigned and synthesized using T1 peptide (SEQ ID NO:36; which has beenreported to be restricted to various MHC class II haplotypes and amultiagretope type T cell epitope derived from HIV IIIB gp120, reportedby Ahlers J. D., Proceedings of the National Academy of Sciences USA,Vol. 94, No. 20, pp. 10856-10861, (1997)) as T cell epitope and OVApeptide fragment (hereinafter, abbreviated as “OVAp”) consisting of 14amino acid residues (SEQ ID NO:38; which is previously disclosed toinduce the production of antibodies specific to ovalbumin, abbreviatedas “OVA”) for BALB/c mouse, reported by Hunt D. F., Science, Vol. 256,pp. 1817-1820, (1992). “OMP-KK-OVAp” (SEQ ID NO:40) and “T1-KK-UP” (SEQID NO:41) were prepared as controls. BALB/c mice were intranasallyimmunized with each above polypeptide according Experiment 6. The resultis shown in Table 9.

TABLE 9 Polypeptide Titer Titer against Titer used as an against theimmunized against Titer against antibody OMP peptide OVA CT OMP-KK-OVAp108 1,589 1,656 — RGD-OMP-KK-OVAp 235 5,080 11,692 — CT + OMP-KK-OVAp7,319 21,310 25,654 3,656,835 Polypeptide Titer Titer against Titer usedas an against the immunized against Titer against antibody T1 peptidePac CT T1-KK-UP 103 160 320 — T1-RGD-KK-UP 107 1,707 1,579 — CT +T1-KK-UP 69 1,152 176 139,264 *“—”: Not tested

As shown in Table 9, the mice immunized intranasally withRGD-OMP-KK-OVAp or T1-RGD-KK-UP showed a remarkable enhancement of theproduction of antibodies specific to OVA or PAc. It is confirmed thatinsertion of RGD sequence imparted the polypeptide to more stronglyenhance the production of antibodies than polypeptides having no RGDsequence.

Experiment 9

Effect of the Polypeptide of the Present Invention on Enhancement of theProduction of Antibodies Specific to Other Antigen

CT, usually used as an adjuvant, is known to strongly induce antibodiesspecific to not only CT but also other antigens when permucosallyadministered together with the other antigens. Otherwise, thepolypeptide of the present invention such as RGD-OMP-KK-UP,OMP-RGD-KK-UP, RGD-OMP-KK-OVAp, and T1-RGD-KK-UP induced specificantibodies even without CT. Following experiment is to investigatewhether the polypeptide enhances the production of antibodies specificto other antigens or not.

Influence of Various Polypeptides for the Production of AntibodiesSpecific to Bovine Serum Albumin in Intranasal Administration

BALB/c mice were immunized with a physiological saline solutioncontaining 4 μg of bovine serum albumin (hereinafter, abbreviated as“BSA”) and either of 1 μl of DUP, RGD-DUP, OMP-KK-UP, OMP-RGD-KK-UP,OMP-KK-OVAp or RGD-OMP-KK-OVAp, or 2 μg of CT in intranasaladministration according to Experiment 6, and titers in the blood weremeasured. The result is shown in Table 10. Titers of antibodies againstBSA, co-administered peptides, and CT were measured.

Influence of Various Polypeptides for Antibody Production AgainstOvalbumin in Intranasal Administration

B10.D2 or BALB/c mice were immunized with a physiological salinesolution containing 4 μg of OVA and either of 1 μg of OMP-RGD-KK-UP,OMP-RGD-KK-OVAp, or 2 μg of CT in intranasal administration according toExperiment 6, and then, titers in the blood were measured. The result isshown in Table 10. Titers of antibodies against OVA, OMP-RGD-KK-OVAp,and CT were measured.

TABLE 10 Titer against Titer against the immunized Polypeptide used asan antibody CT peptide Titer against BSA BSA — 32 — CT + BSA 1,887,437838,861 DUP + BSA — 179 83 RGD-DUP + BSA 6,912 154 RGDS + RGD-DUP + BSA576 128 OMP-KK-UP + BSA 20,480 1,152 OMP-RGD-KK-UP + BSA 109,227 213OMP-KK-OVAp + BSA 1,441,792 224 RGD-OMP-KK-OVAp + BSA 1,527,887 256Titer against OVA B10.D2 OVA — 203 — mouse CT + OVA 681,574 24,576OMP-RGD-KK-UP + OVA — 10,242 24 BALB/C OVA 16 — mouse CT + OVA 524,2884,096 OMP-RGD-KK-UP + OVA — 2,176 26 *“—”: Not tested

As shown in Table 10, antibodies specific to BSA or OVA were hardlyinduced in the blood of the mice when intranasally administering BSA orOVA alone. In contrast, mice immunized with BSA or OVA in combinationwith OMP-RGD-KK-UP, OMP-KK-OVAp or RGD-OMP-KK-OVAp showed a strongenhancement of the production of antibodies specific to BSA or OVA. Inaddition, mice immunized with BSA in combination with RGD-DUP orOMP-KK-UP showed the production of antibodies specific to BSA than.However, latter mice showed lower enhancement than former mice. Miceimmunized with BSA or OVA in combination with CT showed the same degreeof the production to the above mice. Mice immunized in combination with1 μg of OMP-RGD-KK-UP, OMP-KK-OVAp, or RGD-OMP-KK-OVAp showed a slightproduction of antibodies specific to the peptide, in contrast, miceimmunized in combination with 2 μg of CT showed strongly enhancement ofthe production of antibodies specific to CT. Therefore, OMP-RGD-KK-UP,OMP-KK-OVAp, or RGD-OMP-KK-OVAp is confirmed to be useful for inducingantibodies specific to other antigens as an immunological adjuvant inthe manner of using them in an insufficient or small amount to be inducethe production of their antibodies.

As described above, it is confirmed that cell attachment motifs areallowed the polypeptide consisting of B cell epitope connected to T cellepitope with protease recognition site such as KK to enhance antibodyproduction against B cell epitope. The polypeptide of the presentinvention is useful as inducers for specific antibody production orenhancer for antibody production because it is efficiently capable ofinducing antibody production against B cell epitope peptide in thepolypeptide except inducing useless antibody production against othersite than B cell epitope even when administered without immunologicaladjuvant intranasally. As the result that living bodies administeredwith the polypeptide efficiently produce antibody against antigenicprotein deriving the B cell epitope used for immunizing, the polypeptideof the present invention is capable of inducing various antibody such asantibody for preventing infectious, antibody for neutralizing toxin orenzyme active center, and antibody against allergen in the presence ofno immunological adjuvant in mucosal immunization including nasalimmunization. The polypeptide is useful as peptide vaccine forintranasally immunizing in order to prevent infection. The polypeptideeffects immunological adjuvant when administered with other antigentogether.

The following examples explain the polypeptide of the present inventionconcretely, but the present invention must not be restricted by theseexamples.

EXAMPLE 1

Composition for Enhancing the Production of Antibodies Specific to PAc

The syrup agent containing the polypeptide prepared in Experiment 7 wasobtained by dissolving 100 μg/ml of any one of RGD-OMP-KK-UP,RGD-OMP-KK-UP, RGD-OMP-KK-UP, or OMP-KK-UP, and 40% of α,α-trehalose(reagent grade, commercialized by Hayashibara Biochemical Laboratories,Inc., Okayama, Japan) in distilled water, sterilizing by a usual method.The resulting syrup was placed by 2 ml into sterilized vials, andsealed. Since the product is stable and restricted by various MHC classII haplotypes, it exerts the effect of vaccine which enhances to producethe antibody having the effect on preventing dental caries. The productcan also be useful as immunological adjuvant which enhances the antibodyagainst other antigen when permucosally and intracutaneouslyadministered with the polypeptide together.

EXAMPLE 2

Composition for Enhancing the Production of Antibodies Specific to PAc

Agent containing the polypeptide prepared in Experiment 7 was obtainedby dissolving 10 μg/ml, 100 μg/ml or 1,000 μg/ml of RGD-T1-KK-UP inphysiological saline containing 1% (w/v) of sucrose, and sterilizing byfiltration. The resulting solution was placed by 1 ml into sterilizedvials, freeze-dried, and sealed in a usual manner. The product is apreparation for permucosal administration or infection, which is stableand has a satisfactory effect on preventing dental caries. The productis used after dissolved in 1 ml of distilled water for injection. Sincethe product is stable and restricted by various MHC class II haplotypes,it exerts the effect of vaccine, which efficiently enhances theproduction of the antibodies having the effect on the prevention ofdental caries, in intranasal or oral administration for many human oranimals. The product can also be useful as an immunological adjuvantwhich enhances the production of other antibodies specific to otherantigens when permucosally and intracutaneously administered togetherwith the other antigens.

EXAMPLE 3

Toxicity Test for Composition Containing the Polypeptide

LD₅₀ of the preparation containing the polypeptide, prepared in Example1 or 2, was investigated by administering five week-aged DDY male micewith the physiological saline containing 12.5 mg/ml of any one of thepolypeptides and 0.5% of sucrose orally, intraperitoneally, orintramuscularly according to a usual method. As a result, both of theLD₅₀ of the preparations were 100 mg (peptide weight)/kg mouse bodyweight or over. Therefore, the preparations are considered as safepreparations with no toxicity when administered to humans or animals.

EXAMPLE 4

Composition for Enhancing the Production of Antibodies Specific to HIV

Any one of OMP, T1, gag protein at the position of 298-312 (SEQ IDNO:42) and pol protein at the position of 596-610 (SEQ ID NO:43) wasselected as a T cell epitope sequence. The gag protein and pol proteinwere reported as T cell epitopes restricted by HLA-DR1-9 or 51-53, byWilson, C. C., Journal of Virology, Vol. 75, pp. 4195-4207, (2001). Thepolypeptides of the present invention were designed in a manner ofarranging RGD sequence at N-terminal or C-terminal region of either ofthe T cell epitope sequences, and then, arranging B cell epitopesequence of V3 loop peptide of gp120 protein from HIV (SEQ ID NO:44),reported by Haynes, B. F., The Journal of Immunology, Vol. 151, pp.1646-1653, (1993), at the C-terminal region of the resulting sequence,which positions via KK linker inserted between the two sequences. 150μg/ml of the one or two of the above polypeptides and 100 mg/ml ofmannitol were dissolved in physiological saline. The resulting solutionswere placed by 1 ml in 5 ml-volume vials and freeze-dried. The productis useful as a vaccine for inhibiting the spreading of HIV and delayingthe development of HIV because it is capable of efficiently enhancingthe production of antibodies specific to HIV in permucosal orpercutaneous administration. In addition, it enables to target forvariety person because of restricted to various MHC class II haplotypes.The product is also useful as immunological adjuvant for enhancing theproduction of other antibodies specific to other antigens which ispermucosally administered together with the other antigens.

EXAMPLE 5

Composition for Enhancing Antibody Production Against Influenza Virus

The polypeptides of the present invention were designed in a manner ofarranging RGD or YIGSR sequence at N-terminal or C-terminal region ofthe T cell epitope sequence selected from OMP, T1, SEQ ID NO:42 and 43,and then, in order to induce neutralizing antibodies against aninfluenza virus, arranging B cell epitope sequence of HA (hemaggrutinin)protein at the position of 91-108 from influenza virus (SEQ ID NO:45)reported by Ben-Yedidia T., Molecular Immunology, Vol. 39, pp. 323-331,(2002) at the C-terminal region of the resulting sequence, whichpositions via KK linker inserted between the two sequences. Each 75μg/mL of one or more the above peptides was admixed with physiologicalsaline containing 0.5 mg/ml of human albumin. The resulting solutionswere placed by 1 ml in 5 ml-volume vials and freeze-dried in a usualmanner. The product is useful as a vaccine for inhibiting the infectionof influenza virus because it is capable of efficiently enhancing theproduction of the antibodies specific to influenza virus in permucosalor percutaneous administration. In addition, it is enabling to targetfor variety person because of restricted to various MHC class IIhaplotypes. The product is also useful as immunological adjuvant forenhancing the production of other antibodies specific to other antigens,when applied together with the other antigens in permucosaladministration.

EXAMPLE 6

Papilloma Virus Vaccine

The polypeptides of the present invention were designed in a manner ofarranging RGD sequence at N-terminal or C-terminal region of the T cellepitope sequence selected from OMP, T1, SEQ ID NO:42 and 43, and then,in order to induce neutralizing antibodies against human papillomavirus, arranging B cell epitope sequence of L2 protein (SEQ ID NO:45)from human papilloma (SEQ ID NO:46), reported by Kawana K., Vaccine,Vol. 19, pp. 1496-1502, (2001), at the C-terminal region of theresulting sequence, which positions via KK linker inserted between thetwo sequences. Each 200 μg/ml of one or more the above peptides wasadmixed with phosphate buffer saline containing 0.25 mg/ml of gelatin.The resulting solution was placed by 0.5 ml in 5 ml-volume vials andfreeze-dried in a usual manner. The product is useful as a vaccine forinhibiting the infection of human papilloma virus because it is capableof efficiently enhancing the production of antibodies specific to humanpapilloma virus in permucosal or percutaneous administration. Inaddition, it enables to target for variety person because of restrictedto various MHC class II haplotypes. The product is also useful asimmunological adjuvant for enhancing the production of other antibodiesspecific to other antigens when applied together with the other antigensin permucosal administration.

EXAMPLE 7

Composition for the Production of Antibodies Specific to OVA

100 μg/ml of RGD-OMP-KK-OVAp prepared in Experiment 8 was dissolved with40% of α,α-trehalose (reagent grade, commercialized by HayashibaraBiochemical Laboratories, Inc., Okayama, Japan) in distilled water andsterilized in a usual manner. The resulting syrup was placed by 2 ml insterilized vials, and sealed to obtain a syrupy vaccine containing thepolypeptide. The product is satisfactorily stable, and efficientlyenhances the production of the antibodies specific to OVA whenintranasally, orally or percutaneously administered to animals. Theproduct can also be useful as immunological adjuvant which enhances theantibody against other antigen when intranasally administered with thepolypeptide together.

EXAMPLE 8

Composition for Enhancing the Effect of Influenza Vaccine on theProduction of Antibodies

4 μg of either commercialized influenza HA vaccine by Astellas PharmaInc., Tokyo, Japan, or virus surface antigenic protein such as HA and Mprotein was dissolved with 1 μg of either RGD-OMP-KK-OVAp orRGD-OMP-KK-UP in 1 ml of physiological saline. The resulting solutionswere placed by 0.5 ml in sterilized vials and sealed to obtainpharmaceutical preparations. The product containing the usual influenzavaccine or the viral surface antigenic protein, which dose not induce bypermucosal administration in single use, strongly induces the productionof antibodies specific to the viral surface antigenic protein due to theimmunological adjuvant-like action of RGD-OMP-KK-OVAp or RGD-OMP-KK-UP.The product can be used as a vaccine, which strongly induces theproduction of the antibody having an activity for preventing theinfection of influenza virus when intranasally administered three orfour times at two weeks interval.

EXAMPLE 9

Oral Composition for Hyposensitization of Japanese Cedar Pollinosis

The polypeptides of the present invention were designed in a manner ofarranging RGD sequence at N-terminal or C-terminal region of the T cellepitope sequence selected from OMP, T1, SEQ ID NO:42 and 43, and then,in order to induce the production of antibodies specific to Japanesecedar pollen allergens, arranging B cell epitope sequences “VHPQDGDA” ofCry j 1, reported by Kawana K., Vaccine, Vol. 19, pp. 1496-1502, (2001)and “KWVNGRI” of Cry j 2, reported by Tamura Y., Clinical andExperimental Allergy, Vol. 33, No. 2, pp. 211-217, (2003), at theC-terminal region of the resulting sequence, which positions via KKlinker inserted between the two sequences to obtain the polypeptideconsisting of the amino acid sequence of SEQ ID NO:47. Each 200 μg/ml ofone or more the above peptides was admixed with phosphate buffer salinecontaining 0.25 mg/ml of gelatin to obtain an oral composition forhyposensitization of Japanese cedar pollinosis. The product is useful asa preparation for hyposensitization of Japanese cedar pollinosis becauseit is capable of efficiently enhancing the production of IgG antibodiesspecific to Cry j 1 and Cry j 2 in permucosal or percutaneousadministration. In addition, it enables to target for variety personbecause of restricted to various MHC class II haplotypes.

INDUCTRIAL APPLICABILITY

As described above, the present invention relates to the polypeptidecontaining a special antigen peptide, which enables to enhance theproduction of an antibody specific to antigenic epitope of an objectiveantigen or a composition thereof without immunological adjuvant. Thepolypeptide of the present invention is useful as vaccine in use formany person as well as antibody productive animals such as mammals,fowls, reptiles, and fishes, or as antigen for enhancement of specificantibody production because it is capable of using in permucosaladministration such as intranasal and oral administration more easy andsafe than percutaneous injection and is restricted in varieties of MHCclass II haplotypes. It is also useful as immunological adjuvant forenhancing the production of antibodies specific to other antigens whenadministered together with the other antigens.

1. A polypeptide, consisting of the amino acid sequence of SEQ ID NO:32,SEQ ID NO:33 or SEQ ID NO:39.
 2. A composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable additive.